Studies on the transformation of nitrosugars into branched chain iminosugars. Part II: Synthesis of (3R,4R,5R,6S)-2,2-bis(hydroxymethyl)azepane-3,4,5,6-tetraol

Article abstract of DOI:10.1016/j.tetasy.2008.10.027

The first stereocontrolled synthesis of (3R,4R,5R,6S)-2,2-bis-(hydroxymethyl)azepane-3,4,5,6-tetraol is described herein. The method involves a novel double Henry reaction of the 3,5-di-O-benzyl-6-deoxy-1,2-O-isopropylidene-6-nitro-α-d-glucofuranose with formaldehyde followed by a reductive ring closure to give the first branched 1,6-dideoxy-1,6-heptitol described.

Full text of DOI:10.1016/j.tetasy.2008.10.027

Tetrahedron: Asymmetry 19 (2008) 2443–2446
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Studies on the transformation of nitrosugars into branched chain
iminosugars. Part II: Synthesis of (3R,4R,5R,6S)-2,2-bis(hydroxymethyl)azepane-
3,4,5,6-tetraol
José M. Otero a, Amalia M. Estévez a, Raquel G. Soengas a, Juan C. Estévez a, Robert J. Nash b,
George W. J. Fleet c, Ramón J. Estévez a,
*
a Departamento de Química Orgánica, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain
b Summit (Wales) Limited, Plas Gogerddan, Aberystwyth, Coredigon, UK
c Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Mansfield Road, Oxford, OX1 3TA, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The first stereocontrolled synthesis of (3R,4R,5R,6S)-2,2-bis-(hydroxymethyl)azepane-3,4,5,6-tetraol is
described herein. The method involves a novel double Henry reaction of the 3,5-di-O-benzyl-6-deoxy-
1,2-O-isopropylidene-6-nitro-a-D-glucofuranose with formaldehyde followed by a reductive ring closure
Received 22 July 2008
Revised 21 October 2008
Accepted 28 October 2008
to give the first branched 1,6-dideoxy-1,6-heptitol described.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
H
H
N
N
HO
HO
R1
R2
Glycosidase inhibitors have been extensively investigated over
the last two decades due to their potential as therapeutic agents.1
These compounds are involved in several important biological pro-
cesses, such as digestion, biosynthesis of glycoproteins, and catab-
olism of glycoconjugates; some examples have already been tested
or approved for use in the treatment of various diseases, such as
Gaucher’s disease,2 diabetes,3 cancer,4 and viral infections,5 includ-
ing AIDS.6
HO
R
R3
R4
HO
OH
HO
1
3
2
4
3a:
3b:
1: R=H
R=OH
R =R =H; R =R =OH
2
4
1
3
2:
R =R =H; R =R =OH
The most common strategy for the design of such compounds is
based on the quest for mimics of the cyclic alkoxycarbenium tran-
sition state formed during glycosidic bond cleavage. Thus, imino
sugars,7 which are analogs of furanoses or pyranoses where the
ring oxygen is replaced by nitrogen, have been reported in most
cases to be almost always inhibitors of the corresponding glycosi-
dases.8 A great deal of synthetic effort has been focussed on the
preparation of substituted pyrrolidine or piperidine mimics of
the corresponding sugars.9 However, only a few syntheses of high-
er homologues such as azepanes or azocanes have been reported to
date, despite such structures being more flexible than the corre-
sponding pyrrolidines and piperidines and their potential to adapt
better to the active site of glycosidases.
HO
H
H
N
N
HO
HO
HO
OH
HO
OH
OH
OH
HO
4
5
Figure 1.
In 2004, Sinaÿ et al. reported the preparation and biological
evaluation of a number of 1,6-dideoxy-1,6-iminoheptitols 3, a
novel family of polyhydroxylated azepanes that are higher homo-
logues of deoxynojirimycin 4 (Fig. 1).12 Some of these derivatives
show potent and specific glycosidase inhibition; for example,
compound 3a is a potent and selective inhibitor of coffee bean
Tri and tetrahydroxylated azepanes 1 and 2 (Fig. 1) were pre-
pared10 for the first time by Paulsen and Todt in 1967, and have
been extensively studied since then.11 Some of these derivatives
are glycosidase or protease inhibitors, while others exhibited anti-
cancer activity.
a
-galactosidase, and compound 3b strongly inhibits bovine liver
b-galactosidase. On the other hand, due to the unusual spatial dis-
tribution of the hydroxyl groups,13 these azepanes not only display
a different inhibition profile compared to the previously reported
* Corresponding author. Tel.: +34 981 563100; fax: +34 981 591014.
E-mail address: ramon.estevez@si.usc.es (R.J. Estévez).
0957-4166/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2008.10.027

2444
J. M. Otero et al. / Tetrahedron: Asymmetry 19 (2008) 2443–2446
polyhydroxylated azepanes but also they are more prone to form
hydrogen bonds with nitrogenated bases, thus improving their
ability to bind to the minor groove of DNA.14
tionally supported from its stability in methanol at neutral pH
and from that a C-2 epimerization occurred when it was left stand-
ing in a methanolic hydrochloric acid solution. This can be ex-
Derivatives of 1,6-dideoxy-1,6-iminoheptitol provide an oppor-
tunity to alter and hopefully increase the specificity of inhibition of
individual glycosidases. Accordingly, it should be of interest to pre-
pare families of branched 1,6-dideoxy-1,6-iminoheptitols 5 (Fig. 1)
in order to test their activity against a range of glycosidases and to
determine whether the introduction of a branch can alter the bio-
logical activity. As a continuation of work on new synthetic appli-
cations of nitrosugars15 and on new synthetic approaches to
branched iminosugars,16 we herein report the first enantiospecific
synthesis of azepane 5a. The synthesis includes the early introduc-
tion of the two hydroxymethyl substituents at C-6 of nitrosugar 6
by means of a double Henry reaction with formaldehyde (Scheme
1), followed by a heteroannulation strategy that was previously
used by Fleet et al. in the synthesis of six-membered ring
iminosugars.17
plained in terms of
a
dehydration–hydration equilibrium
between compound 10 and its non-isolable derivative 11.
Finally, treatment of 10 with sodium cyanoborohydride affor-
ded the hydrochloride salt of azepane derivative 5a in good yield,
through imine 11.
The new iminoheptitols 10 and 5a were assayed for their inhib-
itory activity toward commercially available glycosidases. Signifi-
cant inhibition of the following glycosidases was not observed at
a concentration of 143
from Saccharomyces cerevisiae,
thermophilus, -glucosidase from rice (Oryza sativa), b-
dase from Almond (Prunus sp.), -galactosidase from green
coffee bean (Coffea sp.), b- -galactosidase from bovine liver,
fucosidase from bovine kidney, -mannosidase from jack bean
(Canavalia ensiformis), b- -mannosidase from Cellullomonas fimi,
naringinase from Penicillium decumbens, N-acetyl-b- -glucosamin-
idase from bovine kidney, N-acetyl-b- -glucosaminidase from jack
bean, N-acetyl-b- -hexosaminidase from Aspergillus oryzae, and
l
g/mL and at optimal pH:
-glucosidase from Bacillus stero-
-glucosi-
a-D-glucosidase
a-D
a
-D
D
a-D
D
a-L-
a-D
D
D
D
2. Results and discussion
D
amyloglucosidase from Aspergillus niger.
The Henry reaction of nitrosugar 6 with paraformaldehyde as a
source of the hydroxymethyl groups and tetrabutylammonium
fluoride as a base afforded dihydroxymethylnitroderivative 7 in
good yield. Treatment of 7 with ammonium formate and a catalytic
amount of palladium black at 50 °C for 24 h gave amino derivative
8. Removal of the isopropylidene protecting group in compound 8
under acidic conditions gave a 1.0:0.4 anomeric mixture of amines
9a and 9b, after the trifluoroacetic acid present in the reaction mix-
ture was co-evaporated with toluene under reduced pressure. The
resulting reaction crude remained unaltered in a sodium bicarbon-
ate solution at room temperature, but compound 10 was obtained
when heated at 40 °C for 24 h. The 13C NMR spectrum of this com-
pound showed that only one species was present, and its 1H NMR
spectrum showed a coupling constant between the protons H-2
and H-3 (J2,3 = 1.2 Hz). This implies a cis relationship between the
anomeric and adjacent hydroxyl groups. The presence of the N
bridge in the azepane ring was confirmed by means of an HMBC
experiment that showed a signal corresponding to the H-2–C-7
long distance coupling. The structure of compound 10 was addi-
3. Conclusion
In conclusion, we have achieved for the first time the efficient
preparation of seven-membered ring branched iminoheptitols
using a double Henry reaction with formaldehyde as the key step.
Our polyhydroxyazepanes have an extra hydroxymethyl group
compared to the previously reported 1,6-dideoxy-1,6-
iminoheptitols.
Inhibition studies on the branched derivative 5a showed that
the introduction of a second hydroxymethyl substituent at the C-
6 position of 3a or 3b resulted in the loss of inhibitory activity. This
is probably due to the conformational distortion caused by the
branched substituent as compared to 3a and 3b, which results in
5a being unable to efficiently bind to the active site of any of the
above glycosidases.
Work is now currently in progress in order to extend these
studies to hexoses other than
D-glucose in order to gain access to
HO
HO
OH
O
OH
O2N
O2N
H2N
HO
i
ii
O
BnO
O
O
BnO
O
O
O
BnO
O
O
BnO
HO
8
6
7
iii
HO
OH
O
.
N
HO
HO
HO
HO
HO
HO
H HCl
H
N
OH
H2N
HO
N
v
iv
HO
HO
HO
OH
OH
OH
OH
OH
OH
OH
HO
HO
HO
OH
HO
9a
5a
11
10
:
9b:
Scheme 1. Reagents and conditions: (i) (HCHO)n, TBAF, THF, rt, 1 h (86%); (ii) Pd black, NH4HCO2, MeOH, 50 °C, 24 h (88%); (iii) TFA, H2O, rt, 4 h; (iv) NaHCO3, THF, 40 °C, 24 h
(83%); (v) (1) NaCNBH3, AcOH, MeOH, rt, 30 h; (2) AcCl, MeOH, rt, 30 min (78%).

J. M. Otero et al. / Tetrahedron: Asymmetry 19 (2008) 2443–2446
2445
a wide range of seven-membered ring branched iminosugars like
5a. Inhibition studies on these targets together with conforma-
tional and spectroscopical studies will allow us to confirm the
above hypothesis on the relationship between the conformational
properties of compounds 5 and the absence of glycosidase inhibi-
tion properties.
d = 1.31 (s, 3H, CH3); 1.47 (s, 3H, CH3); 3.74 (d, 1H, J7,7 = 11.5 Hz,
H-7); 3.76 (d, 1H, J7,7 = 11.5 Hz, H-7); 3.86 (d, 1H, J7,7 = 11.5 Hz,
H-7); 3.91 (d, 1H, J7,7 = 11.5 Hz, H-7); 4.14 (d, 1H, J3,4 = 10.3 Hz,
H-3); 4.22–4.28 (m, 2H, H-4 + H-5); 4.48 (d, 1H, J1,2 = 3.6 Hz, H-
2); 5.92 (d, 1H, J1,2 = 3.6 Hz, H-1); 8.51 (br s, 2H, NH2). 13C NMR
(CD3OD) d = 26.7; 27.3; 61.1; 64.2; 66.9; 75.6; 81.2; 85.5; 106.8;
113.1. IR (NaCl)
m = 3425 (br, OH + NH2). MS (CI) m/z (%) = 280
(100, MH+); 263 (46, [MꢂNH2]+); 222 (27, [MꢂC3H6O]+).
4. Experimental
4.3. 6-Amino-6-deoxy-6-C-hydroxymethyl-
heptofuranose 9a and 6-amino-6-deoxy-6-C-hydroxymethyl-b-
a-D-gluco-
Melting points were determined using a Kofler Thermogerate
apparatus and are uncorrected. Specific rotations were recorded
on a JASCO DIP-370 optical polarimeter. Infrared spectra were re-
corded on a MIDAC Prospect-IR spectrophotometer. Nuclear mag-
netic resonance spectra were recorded on a Bruker DPX-250
apparatus. Mass spectra were obtained on a Hewlett Packard
5988A mass spectrometer. Elemental analyses results were ob-
tained from the Elemental Analysis Service at the University of
Santiago de Compostela. Thin layer chromatography (TLC) was per-
formed using Merck GF-254 type 60 silica gel and ethyl acetate/
hexane mixtures as eluants; the TLC spots were visualized with
Hanessian mixture. Column chromatography was carried out using
Merck type 9385 silica gel.
D-gluco-heptofuranose 9b
A solution of 6-amino-6-deoxy-6-C-hydroxymethyl-1,2-O-iso-
propylidene-
D
-glycero-
a
-D
-gluco-heptofuranose
8
(0.17 g,
0.61 mmol) in a mixture of trifluoroacetic acid and water (2:1,
9 mL) was stirred at room temperature for 4 h. The solvents were
removed in vacuo, and the residue co-evaporated with toluene
(3 ꢀ 5 mL) to afford a crude yellow oil formed for a 1.0:0.4 anomer-
ic mixture of 6-amino-6-deoxy-6-C-hydroxymethyl-a-D-gluco-
heptofuranose 9a and 6-amino-6-deoxy-6-C-hydroxymethyl-b-D-
gluco-heptofuranose 9b (0.15 g, 0.61 mmol). 1H NMR (CD3OD)
d = 3.19–3.34 (m, 0.8H, 2 ꢀ 9b-H); 3.34–3.50 (m, 1.4H, 9a-H + 9b-
H); 3.60–3.92 (m, 6.2H, 5 ꢀ 9a-H + 3 ꢀ 9b-H); 3.97–4.13 (m,
2.8H, 2 ꢀ 9a-H + 2 ꢀ 9b-H); 4.57 (d, 0.4H, J1,2 = 7.9 Hz, 9b-H-1);
5.21 (d, 1H, J1,2 = 3.6 Hz, 9a-H-1). 13C NMR (CD3OD) d = 60.9 (9b);
61.0 (9a); 61.1 (9b); 61.2 (9a); 63.3 (9a); 63.4 (9b); 69.2 (9a);
71.8 (9b); 72.0 (9a); 73.0 (9a); 73.6 (9b); 75.0 (9a); 75.7 (9b);
4.1. 3,5-Di-O-benzyl-6-deoxy-6-C-hydroxymethyl-1,2-O-
isopropylidene-6-nitro-a-D-gluco-heptofuranose 7
A 1.0 M solution of tetrabutylammonium fluoride in dry tetra-
hydrofuran (1.34 mL, 1.34 mmol) was added to a suspension of
paraformaldehyde (0.41 g, 13.5 mmol) and 3,5-di-O-benzyl-6-
78.0 (9b); 93.8 (9a); 98.6 (9b). IR (NaCl)
m = 3327 (br, OH + NH2).
MS (CI) m/z (%) = 240 (7, MH+); 223 (4, [MꢂNH2]+); 222 (16,
deoxy-1,2-O-isopropylidene-6-nitro-a-D-glucofuranose 6 (0.29 g,
[MꢂOH]+).
0.67 mmol) in anhydrous tetrahydrofuran (3.5 mL). The reaction
mixture was stirred at room temperature under a nitrogen atmo-
sphere for 1 h. Dichloromethane (10 mL) was added, and the or-
ganic layer was washed with saturated aqueous ammonium
chloride (3 ꢀ 5 mL), dried with anhydrous sodium sulfate, filtered,
and evaporated in vacuo. The resulting crude product was purified
by flash column chromatography (dichloromethane/methanol
50:1) to give 3,5-di-O-benzyl-6-deoxy-6-C-hydroxymethyl-1,2-O-
4.4. (2R,3R,4S,5R,6R)-7,7-Bis-(hydroxymethyl)azepane-
2,3,4,5,6-pentaol 10
Sodium bicarbonate (0.077 g, 0.915 mmol) was added to a solu-
tion of hemiacetals 9a and 9b (0.15 g, 0.61 mmol) in tetrahydrofu-
ran (9 mL), and the resulting solution was heated at 40 °C for 24 h,
after which TLC (chloroform/methanol/water/acetic acid
60:30:5:3) showed that the starting material had been consumed.
The solvent was evaporated in vacuo, and the resulting residue dis-
solved in acetone. The solution was filtered, and the filtrate was
concentrated under reduced pressure. The residue was purified
by flash column chromatography (chloroform/methanol/water
40:10:1) to give (2R,3R,4S,5R,6R)-7,7-bis-(hydroxymethyl)aze-
pane-2,3,4,5,6-pentaol 10 (0.12 g, 0.51 mmol, 83% yield) as a yel-
isopropylidene-6-nitro-
a
-
D
-gluco-heptofuranose
7
(0.29 g,
¼ ꢂ67:8
0.58 mmol, 86% yield) as an amorphous white solid. ½a D20
ꢁ
(c 1.70, CHCl3). 1H NMR (CDCl3) d = 1.32 (s, 3H, CH3); 1.49 (s, 3H,
CH3); 2.72 (br s, 1H, OH); 2.95 (br s, 1H, OH); 4.10 (d, 1H,
J4,5 = 3.1 Hz, H-5); 4.21–4.28 (m, 4H, 2 ꢀ CH2-Ph); 4.37–4.47 (m,
4H, 2 ꢀ CH2–OH); 4.64 (d, 1H, J1,2 = 3.9 Hz, H-2); 4.66–4.71 (m,
2H, H-3 + H-4); 5.91 (d, 1H, J1,2 = 3.9 Hz, H-1); 7.10–7.39 (m, 10H,
10 ꢀ H-Ph). 13C NMR (CDCl3) d = 26.2; 26.7; 62.3; 63.6; 71.2;
75.1; 77.1; 78.5; 80.1; 81.7; 95.9; 104.9; 112.3; 127.3; 127.6;
ꢀ
low oil. ½a 2D0
ꢁ
¼ ꢂ13:9 (c 1.32, CH3OH). 1H NMR (D2O) d = 3.39 (s,
1H, NH); 3.46 (d, 1H, J6,6 = 8.4 Hz, H-6); 3.57 (dd, 1H, J2,3 = 1.2 Hz,
J3,4 = 7.3 Hz, H-3); 3.71 (dd, 1H, J8,8 = 2.3 Hz, J8,8 = 10.4 Hz, H-8);
3.77–3.80 (m, 3H, H-5 + 2 ꢀ H-8); 3.87–3.91 (m, 2H, H-4 + H-8);
4.99 (d, 1H, J2,3 = 1.2 Hz, H-2). 13C NMR (D2O) d = 61.4; 67.0; 69.0;
127.9; 128.2; 128.4; 128.6; 136.6; 137.1. IR (NaCl)
m = 3472 (br,
OH); 1546 (st, NO2). MS (CI) m/z (%) = 491 (1, [M+H2]+); 91 (100,
[PhCH2]+). Anal. Calcd for C25H31NO9: C, 61.34; H, 6.38; N, 2.86.
Found: C, 60.98; H, 6.23; N, 2.82.
69.3; 71.6; 72.2; 77.3; 94.4. IR (NaCl)
m = 3420 (br, OH + NH). MS
(ES) m/z (%) = 240 (100, MH+); 222 (34, [MꢂH2O]+). HRMS calcu-
lated for C8H18NO7 [MH]+: 240.1083. Found: 240.1077,
4.2. 6-Amino-6-deoxy-6-C-hydroxymethyl-1,2-O-
isopropylidene-a-D-gluco-heptofuranose 8
D
m = 6 ꢀ 10ꢂ4
.
Palladium black (0.11 g, 20% w/w) and ammonium formate
(2.09 g, 33.11 mmol) were added to a degassed solution of 3,5-di-
O-benzyl-6-deoxy-6-C-hydroxymethyl-1,2-O-isopropylidene-6-ni-
4.5. (3R,4R,5R,6S)-2,2-Bis-(hydroxymethyl)azepane-3,4,5,6-
tetraol hydrochloride 5a
tro-a-D-gluco-heptofuranose 7 (0.54 g, 1.10 mmol) in methanol
Sodium cyanoborohydride (0.045 g, 0.71 mmol) was added to a
solution of (2R,3R,4S,5R,6R)-7,7-bis-(hydroxymethyl)azepane-
2,3,4,5,6-pentaol 10 (0.034 g, 0.14 mmol) in a mixture of methanol
and acetic acid (98:2, 1 mL). The mixture was stirred at room tem-
perature for 24 h. The solvents were removed in vacuo, after which
the residue was dissolved in anhydrous methanol (1 mL) and acet-
yl chloride (0.1 mL) was added dropwise. The mixture was stirred
(11 mL), and the resulting mixture was stirred under a nitrogen
atmosphere at 50 °C for 24 h. The suspension was then filtered
through a CELITEÒ pad, and the solvent was evaporated in vacuo,
to give 6-amino-6-deoxy-6-C-hydroxymethyl-1,2-O-isopropyli-
dene-a-D-gluco-heptofuranose 8 (0.27 g, 0.97 mmol, 88% yield) as
a yellow oil. ½a 2D0
ꢁ
¼ ꢂ20:3 (c 2.33, CH3OH). 1H NMR (CD3OD)

2446
J. M. Otero et al. / Tetrahedron: Asymmetry 19 (2008) 2443–2446
for 30 min, and the white solid was filtered off and was washed
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hydrochloride
5a
(0.028 g, 0.11 mmol, 78% yield). ½a D20
ꢁ
¼ ꢂ5:6 (c 1.07, CH3OH). 1H
NMR (D2O) d = 3.41–3.58 (m, 3H, 2 ꢀ H-7 + H-6); 3.89–4.18 (m,
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m = 3323 (br, OH + NH).
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calculated for C8H18NO6 [MꢂCl]+: 224.1134. Found: 224.1133,
D
m = 1 ꢀ 10ꢂ4
.
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pH for the enzyme. The incubation mixture consisted of 10
enzyme solution, 10 L of 1 mg/mL aqueous inhibitor solution,
and 50 L of the appropriate 5 mM para-nitrophenyl substrate
made up in buffer at the optimum pH for the enzyme. The reac-
tions were stopped by the addition of 70 L of 0.4 M glycine (pH
lL of
l
l
l
10.4) during the exponential phase of the reaction, which had been
determined at the beginning using uninhibited assays in which
water replaced inhibitor. Final absorbances were read at 405 nm
using a Versamax microplate reader (Molecular Devices). Assays
were carried out by triplicate.
Acknowledgments
We thank the Spanish Ministry of Science and Innovation for
the financial support (CTQ2005-00555) and for F.P.U. grants to
José M. Otero and Amalia Estévez, and the Xunta de Galicia for
the financial support and for a grant to Raquel G. Soengas.
17. Fleet, G. W. J.; Carpenter, N. M.; Petursson, S.; Ramsden, N. G. Tetrahedron Lett.
1990, 31, 409.